Synaptic vesicles are highly specialized organelles located at synaptic nerve terminals which contain chemical neurotransmitters. Depolarization of the nerve terminal and consequent calcium ion influx triggers synaptic vesicle fusion with the presynaptic plasma membrane and exocytosis occurs, releasing neurotransmitters into the synaptic cleft. After exocytosis, the synaptic vesicle membrane proteins are rapidly internalized and used in the reformation of synaptic vesicles at the nerve terminal. The molecular mechanism of synaptic vesicle reformation is not well understood: compelling evidence suggests that synaptic vesicle membrane proteins are present in early endosomes, and from endosomes, the vesicle membranes bud and pinch off to reform the small, uniformly-sized vesicles active in secretion. This proposal describes experiments designed to elucidate the molecular mechanism of synaptic vesicle reformation from endosomes. The rationale underlying the proposed experiments is based upon recent preliminary data generated in the sponsor's laboratory demonstrating that clathrin coated buds possessing dynamin "collars" are present on endosomes in the nerve terminal in the presence of the non-hydrolyzable GTP analogue, GTP-gammaS. Based on these preparations, an in vitro synaptic vesicle budding assay will be developed which will provide a measure of synaptic vesicle formation from endosomes under a variety of conditions. The possible roles of the neuron-specific proteins dynamin-1 and p145, two Grb-2 binding-proteins, which undergo dephosphorylation in response to nerve terminal depolarization, and of clathrin associated protein AP-3, will be examined in detail through the use of biochemical and morphological assays.